Abundant, distinct, and seasonally dynamic bee community in the canopy‐aerosphere interface above a temperate forest

Abstract Our understanding of how bees (Apoidea) use temperate forests is largely limited to sampling the understory and forest floor. Studies over the last decade have demonstrated that bee communities are vertically stratified within forests, yet the ecology of bee assemblages immediately above the canopy, the canopy‐aerosphere interface, remains unexplored. We sampled and compared bee communities above the canopy of a temperate forest to the understory (1 m), midstory (10 m), and canopy (20 m) on the campus of the University of Massachusetts, in Amherst, Massachusetts, United States from April to August, 2021. Overall, we found that assemblages above the canopy had more bees than in the understory, were distinct in composition from all other strata, and included the greatest proportion of unique species. Bee abundance and species richness were highest in the understory throughout the spring (April and May) and decreased as the season progressed, while bee abundance and species richness at higher strata increased into the summer months. We also found that bees with preferences to nest in moist and rotting wood were largely restricted to canopy and midstory strata. We conclude that bee assemblages occupying the space above the forest canopy are abundant and diverse, and their unique composition suggests that this canopy‐aerosphere interface plays an additional role in the bee community of temperate forests. Alternatively, our findings question how forest bee communities should be defined while highlighting the need for research on fundamental processes governing species stratification in and above the canopy.


| INTRODUC TI ON
Studies examining bee communities within temperate forests have largely restricted sampling to the understory  with the presumption that most bees remain in this lower stratum.
However, recent evidence indicates that bees are vertically distributed within temperate forests (e.g., Allen & Davies, 2022;Ulyshen et al., 2010;Urban-Mead et al., 2021), suggesting a potentially large knowledge gap in the ecological role of these important forest pollinators. Despite this revelation, research regarding the vertical distribution of bees and other pollinators within forests is further limited by the difficulty of sampling the high canopy (Cannon et al., 2021;Cunningham-Minnick et al., 2022). Current sampling methods reach into the canopy (e.g., Maguire et al., 2014;Ulyshen et al., 2010), but the canopy-aerosphere interface-a potentially ecologically important area for bees due to copious floral resources available-remains unexplored in temperate forests (Nakamura et al., 2017;Urban-Mead et al., 2021). Thus, our understanding of pollinator ecology within forests will remain incomplete until the distribution of forest bee communities along the entire vertical gradient of vegetation structure is documented. Moreover, if the current understanding of bee abundance and diversity patterns in forests is inaccurate, forest management recommendations for bee conservation may be biased or potentially misguided Urban-Mead et al., 2021), further highlighting the importance of understanding the distribution of bee communities along the full vertical gradient of temperate forests, including the canopy-aerosphere interface.
Bees are expected to be spatially and temporally distributed throughout temperate forests in response to local resource availability. Studies have demonstrated that forest bee communities are diverse and vertically stratified on sun-exposed edges (e.g., Allen & Davies, 2022;Cunningham-Minnick & Crist, 2020) and within the forest interior (e.g., Allen & Davies, 2022;Campbell et al., 2018;Milam et al., 2022;Ulyshen et al., 2010;Urban-Mead et al., 2021) when floral resources of the forest are available, as well as when they are not. Inferences and observations further suggest that bees will both forage on floral resources and nest at different vertical strata within forests (Allen & Davies, 2022;Cunningham-Minnick & Crist, 2020;MacIvor et al., 2014;Russo & Danforth, 2017;Smith et al., 2019;Sobek et al., 2009;Urban-Mead et al., 2021;Wood et al., 2018). For instance, Smith et al. (2019) and Wood et al. (2018) found support through pollen analyses that forest bee communities rely upon floral resources of dominant tree species. Yet floral resources of herbaceous and woody species within temperate forests are typically limited to spring and early summer phenology, which has been correlated to fewer late-season bees in the forest understory (Cunningham-Minnick & Crist, 2020). Alternatively, studies have found more bees in the forest herbaceous layer during spring and more bees in the canopy during the summer (Cunningham-Minnick & Crist, 2020;Ulyshen et al., 2010), suggesting that the distribution of forest bees may also shift out of the understory and into the higher vertical strata of the forest as the year progresses. However, no studies have compared the bee fauna in the aerosphere above the forest canopy to strata within temperate forests. Thus, we undertook this study to determine the extent to which bees occupy the open air above the forest canopy, how the bee assemblages of this canopy-aerosphere interface compare in abundance, species richness, and composition with assemblages at other strata, and how these patterns change with seasonal phenology.

| MATERIAL S AND ME THODS
We selected two trees ⁓50 m apart in each of two forest patches on the campus of the University of Massachusetts-Amherst in Amherst, Mass., USA ( Figure A1); each pair of trees consisted of a northern red oak (Quercus rubra L.) and a red maple (Acer rubrum L.). Both sites were in USDA Hardiness Zone 5a and were characterized by an herbaceous stratum of ferns (e.g., Dennstaedtia punctilobula (Michx.) T.  (Batra, 1985;Cunningham-Minnick et al., 2022).
We chose these forest patches due to their accessibility and general representation of dominant species in forests of the area.
The bee community was sampled using blue vane traps in the understory, midstory, canopy, and above canopy strata of the forests at each focal tree. Three traps were individually attached to a rope hung over a high branch in the canopy as in Cunningham-Minnick and Crist (2020). Traps were placed 1, 10, 20, and ⁓30 m above the ground (Table A1) to represent the following strata: understory, midstory, canopy and above canopy ( Figure 1). The trap above the canopy was set 1 m above the tallest leaf-bearing branch of each tree using a telescoping hanger attached to a vertical limb in the crown of the canopy as described in Cunningham-Minnick et al. (2022). Traps were deployed on April 2, 2022, and checked every 1-3 weeks until August 21, 2022, for a total of 12 checks.
Bees were sorted, pinned, and identified to species by JM using published keys (e.g., Gibbs, 2011;Gibbs et al., 2013;LaBerge, 1987LaBerge, , 1989Mitchell, 1960Mitchell, , 1962 and the online source Disco verli fe.org (Ascher & Pickering, 2020); vouchered specimens are located at the Natural History Collections at the University of Massachusetts in Amherst, MA. To distinguish differences in microclimate from other conditions among strata, Onset HOBO® Pendant data loggers (Part AU-002-64) were placed directly above each trap to record the light intensity and temperature every 10 min from June 7-21, 2022, to provide data on daily microclimate conditions and hourly from June 22-August 21, 2022, to represent seasonal change.

| Data analysis
To compare bee abundance and species richness across vertical strata throughout the sampling season, we built generalized linear mixed effects models with negative binomial errors and created 95% confidence intervals of pairwise comparisons for each response across strata. All analyses were performed in the R statistical software (R Core Team, 2021). Models were made using the glmmTMB function in the glmmTMB package (Brooks et al., 2017) with fixed effects of stratum (understory, midstory, canopy, above canopy), sample (1-12) as a continuous variable, and their interaction. We allowed the model intercept to vary by each unique tree from which traps were hung to account for tree-and location-specific differences. We also included an offset term of the log of the trap deployment duration (days) to account for differences in sampling effort.
The significance of interaction terms was evaluated by likelihood ratio tests; simulated model residuals through the DHARMa package were used to evaluate the overall model fit (Hartig, 2020). Post-hoc comparisons were made using the confint and glht functions in the multcomp package (Hothorn et al., 2008). Differences in bee species composition among strata were visualized with nonmetric multidimensional scaling ordinations performed on a species occurrence matrix of Sorensen distances using the metaMDS function in the vegan package (Oksanen et al., 2019); statistics and p-values were derived using the pairwiseAdonis function with a Bonferroni adjustment for multiple comparisons (Arbizu, 2017). To align our sampling design with the ecological processes of the study area, we considered bees encountered after June 7 as associated with summer conditions. For instance, floral resources were abundant and the canopy was open throughout the vertical gradient of the forest prior to this date but not after (Figure 1).

F I G U R E 1
Sampling trap design and composition (first three letters of genus) for the bee community at 1, 10, 20, and 30 meters above the ground with blue vane traps to represent the understory, midstory, canopy, and above the canopy of the forest, respectively, in the spring when floral resources were available and in the summer at full leaf-out when floral resources were depleted. Augochlorella and Anthidium genera are unabbreviated to differentiate from Augochlora (Aug) and Anthidiellum (Ant), respectively. Traps in the understory, midstory, and canopy were attached to a rope hung over a high branch in the canopy and anchored to a nearby stem for easy collection. The trap above the canopy was employed using a telescoping hanger designed as described in Cunningham-Minnick et al. (2022), which had a rope threaded through the hanger that was anchored to the stem in the understory to allow the trap to be lowered along with another cord at the trap to aid in lowering (not depicted). Numbers next to pie charts represent total abundance across sites.

| RE SULTS
We collected 144 bees representing 37 species in the understory, 170 bees from 31 species in the midstory, 198 bees consisting of 36 species in the canopy, and 167 bees from 28 species in the aerosphere above the canopy, for a total of 679 bees representing 75 species across strata (Table A2; full details in Data.xlsx of supporting information). Twelve specimens could not be identified to species due to body damage and were not included in species richness or composition analyses. After accounting for differences among individual trees, generalized linear mixed models found that there were significantly more bees and bee species in the understory than within, or above, the canopy (Figure 2c,f). Interaction terms (abundance: χ 2 (3) = 19.0, p < .0005; richness: χ 2 (3) = 16.4, p < .001) demonstrated that bee abundance (χ 2 (7) = 24.1; p < .005) and species richness (χ 2 (7) = 30.8; p < .0001) changed among strata throughout the study period (Figure 2b,e; Figure A2). Specifically, bee abundance and species richness were highest within the understory during the spring months (April and May) and decreased as the season progressed, while more bees and more species were encountered in and above canopy layers during the summer months (Figures 1 and 2a,d).
Species composition of the bee community above the canopy was significantly different from the understory, midstory, and canopy layers (Figure 1), but there were no statistical differences among the lower strata (Table A3; Figure A3). For instance, the most abundant (>10% relative abundance) bee genera in the spring months above the canopy were Andrena and Apis, while Lasioglossum, Andrena, Osmia, and Ceratina were common of strata within the forest. Similarly, Bombus and Apis were most abundant above the canopy during the summer months, while Bombus, Lasioglossum, Augochlora, and Ceratina bees were commonly encountered in lower strata. It was also found that abundant species were collected across strata, whereas 13 species occurred only above the canopy (Table 1; Figures A4 and A5).

| DISCUSS ION
Our study is one of the first to demonstrate that bees occupy the aerosphere immediately above the canopy in temperate forests; furthermore, the community above the canopy was compositionally distinct with similar abundances compared with lower strata (understory, midstory, canopy). These findings expand our understanding of forest bee communities and build on earlier research that revealed differences between understory and canopy bees (Allen & Davies, 2022;Cunningham-Minnick & Crist, 2020;Milam et al., 2022;Ulyshen et al., 2010;Urban-Mead et al., 2021). However, when attempting to characterize the forest bee community, the importance of sampling F I G U R E 2 Relationships between bee abundance (a-c) or species richness (d-f) and time of year among strata, including mean values of the data (a and d), fitted mixed effects models with 95% CI (b and e), and pairwise contrasts (logged response) between strata (c and f): Above canopy (A), Canopy (C), Midstory (M), and Understory (U). the canopy-aerosphere interface hinges upon the question of whether these bees should be considered as part of the forest community, or if they are transients moving among resources. The fact that the above canopy assemblage was generally characterized by many species that were not observed at lower strata and were also associated with nonforested habitats (e.g., Agapostemon texanus Cresson, Halictus parallelus Say, Eucera pruinosa (Say); Harrison et al., 2018) suggests that while some bees may forage on floral resources available at tree crowns in the spring, many others may be moving over the forest to access other habitat patches or resources throughout the season, as reported in other insect taxa (Wainwright et al., 2017). We report our findings as broad collective patterns of multiple species and caution against making species-specific inferences since this was a pilot study with limited data. Nevertheless, the presence of an abundant and species-rich bee assemblage at the canopy-aerosphere interface, which had not previously been prioritized, suggests that more studies are needed to address the extent to which these bees should be considered a subset of the forest bee community.
Our study also demonstrated how the vertical stratification of rubrum, suggesting that this stratum may provide access to the floral resources of the forest canopy. However, it seems unlikely that summer bees above the canopy were foraging or nesting since there were negligible forest floral resources and most bees were soilnesting species. Vegetation height has been negatively associated with bee abundance and diversity (Roberts et al., 2017); therefore, bees may instead use the canopy-aerosphere interface for movement or dispersal since this space lacks the obstacles created by the vegetation structure of forest interiors. Alternatively, bees may be physiologically driven to take advantage of the greater light intensities and warmer temperatures above the canopy compared with other strata to forage earlier or later in the day ( Figure A6; Kebler & Somanathan, 2019;Roubik, 1993). It is also possible that some species were seeking mates above the canopy. For instance, groups of male Apis mellifera L. mate with females 10-40 m above the ground (Ruttner, 1966); similarly, male groups of some Bombus species will fly to higher elevations to mate with emerging females, a behavior known as "hill-topping" (Goulson et al., 2011). Though A. mellifera and Bombus spp. comprised 56% of the overall abundance of bees above the canopy, mating behaviors are unlikely to explain our findings because only three individuals of these species were males and three were reproductive females (all Bombus). These data also suggest there was no risk of oversampling the important genus of pollinator Bombus through the continuous deployment of blue vane traps in our study design. However, we did not quantify the density of reproductive females in this, or any other, genus in the study area and recognize that there were likely many more bee species within and above the forest canopy that would be revealed with additional trap types (Prendergast et al., 2020). We also terminated sampling at the end of August due to large declines in bee abundance and species richness observed throughout forest strata. Therefore, it is possible that additional patterns associated with common forest bees, which occur later in the year, such as mating of Bombus spp., were not observed. We also found that males of two solitary soil-nesting species, Andrena imitatrix Cresson and A. mandibularis Robertson, comprised 57% of bee abundance above the canopy in the spring. We are not aware of any studies addressing mating behaviors similar to hill-topping in these species or the genus Andrena.
However, Urban-Mead et al. (2022) found that male A. imitatrix consumes pollen of forest species (Urban-Mead et al., 2022), including A. rubrum (personal communication), a tree species over which we encountered 93% of all A. imitatrix males in the dataset. Thus, there are many potential mechanisms that need to be tested to explain the occurrence of each species encountered above the forest canopy.
There were notable differences in bee assemblages among the other strata that may be best explained through life-history traits.
For instance, bees in our study that nest in moist, decayed wood TA B L E 1 Total bee abundance and species richness, as well as number of unique species, females, soil-nesting species, soil-nesting individuals, woodnesting species that prefer moist and decayed ("soft") wood, and soft woodnesting individuals at each stratum (e.g., Augochlora pura (Say), Lasioglossum coeruleum (Robertson), L.
Our findings are consistent with other studies that demonstrated a high abundance of wood-nesting bees within the canopy (e.g., Campbell et al., 2018;Cunningham-Minnick & Crist, 2020;Ulyshen et al., 2010;Urban-Mead et al., 2021) and suggest that bees that nest in wood, including species that nest in moist decayed wood, or "soft" wood, exhibit a preference for canopy strata within forests likely due to the availability of wood-nesting substrates such as dead limbs or knot holes. Available nesting substrate in the canopy has yet to be tested as a mechanism to explain the high abundance of woodnesting bees within the higher strata of forests since there is welldocumented availability of dead and rotting wood on the forest floor.
Yet, there is a lack of correlation between coarse woody debris on the ground and the abundance of this guild in the canopy (Campbell et al., 2018;Ulyshen et al., 2010;Urban-Mead et al., 2021). On the other hand, the hypothesis that native bees of temperate forests are abundant in the canopy strata during the summer due to the availability of alternative food sources has not been addressed either (Campbell et al., 2018;Ulyshen et al., 2010). Therefore, studies that quantify potential nesting substrates and alternative food sources for wood-nesting bees within the canopy, including those that nest in "soft" wood, are clearly needed to resolve these discrepancies (Harmon-Threatt, 2020). Milam et al. (2022) found that the inclusion of canopy sampling in addition to understory sampling did not influence their ability to characterize the forest bee community. Our study supports their conclusion when only considering bees below the maximum height of the canopy (i.e., understory, midstory, and canopy strata) but further demonstrates that the bee community above the canopy is distinct from lower strata.

ACK N OWLED G M ENTS
We first recognize that our study area sits on the ancestral land of the Pocumtuc people. We thank the University of Massachusetts-Amherst, as well as Todd Cournoyer, campus grounds manager, and Brady Yacek, campus arborist, for their support and permission to use woodlots on campus.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly avail-

F I G U R E A 1
Aerial photo of the two forest fragments within the study area. Yellow icons mark locations of sites. The bee community was sampled at one Acer rubrum and one Quercus rubra tree within each site.

TA B L E A 3
Test statistic (Pseudo-F) on one degree of freedom from simulated contrasts of species composition between strata with associated p-value adjusted for multiple comparisons.

F I G U R E A 3
Nonmetric multidimensional scaling of bee community composition at each stratum, marked by colored dots and 95% confidence ellipses. Black dots represent species scores.

F I G U R E A 4
Stacked bar plot of bee abundances among forest strata for all bee species with >5 total individuals collected.

F I G U R E A 5
Spring (April 2-June 7) and summer (June 8-August 21) bee community composition at 1 m (Understory), 10 m (Midstory), 20 m (Canopy), and 30 m (Above canopy) above the forest floor. Number in parentheses represents total abundance. Species in overlapping ellipses were at multiple strata.

F I G U R E A 6
Generalized additive regressions of mean temperatures (a, b) and light intensities (c, d) of traps at each stratum with 95% CI. Readings were recorded in 60-min intervals from June 22 to August 21 (a, c). Records were taken at 10-min intervals (b, d) from June 7 to 21 to represent a typical 24-h (x-axis) day.